s2fieldsofstudy: Biology Chemistry

text:
Design of Antimicrobial Peptides with Cell-Selective Activity and Membrane-Acting Mechanism against Drug-Resistant Bacteria

Antimicrobial peptides (AMPs) can combat drug-resistant bacteria with their unique membrane-disruptive mechanisms. This study aimed to investigate the antibacterial effects of several membrane-acting peptides with amphipathic structures and positional alterations of two tryptophan residues. The synthetic peptides exhibited potent antibacterial activities in a length-dependent manner against various pathogenic drug-resistant and susceptible bacteria. In particular, the location of tryptophan near the N-terminus of AMPs simultaneously increases their antibacterial activity and toxicity. Furthermore, the growth inhibition mechanisms of these newly designed peptides involve cell penetration and destabilization of the cell membrane. These findings provide new insights into the design of peptides as antimicrobial agents and suggest that these peptides can be used as substitutes for conventional antibiotics.

Introduction
Overuse and improper application of antibiotics in the biomedical field has led to the emergence of multidrug-resistant bacteria [1][2][3], making it critical to develop a new generation of antimicrobial agents to replace those that are no longer effective. Several studies have been conducted to identify novel antibiotics from various natural sources [4][5][6]. Antimicrobial peptides (AMPs) are promising such candidates, being host defense molecules that [7][8][9][10][11][12] serve as the initial line of immunological protection against microbial infection in several animals. Although the secondary structures of AMPs are classified into α-helices, β-sheets, extended, and flexible loop structures [13,14], they have common features such as a broad antibacterial spectrum, amphipathic properties, short amino acid length, cationic net charge, and rapid killing kinetics [7][8][9][10][11][12]. Furthermore, the low levels of resistance-inducing behavior of AMPs make them superior to small-molecule antibiotics [12]. Membranolytic AMPs usually cause potential membrane changes or direct membrane disruption via four distinct mechanisms: (1) barrel stave, (2) carpet, (3) toroidal, and (4) aggregation [15][16][17][18][19][20]. Other AMPs enter the cytoplasm of microbial cells via spontaneous lipid-assisted and receptoror channel-mediated translocation. Several mechanisms including transient or disordered pore formation and lipid phase alteration enable AMP penetration [21,22]. However, the AMP uptake mechanisms of many bacteria-penetrating peptides and their cytoplasmic targets have not yet been elucidated.
Previous studies have reported that amphipathic α-helix structures with hydrophobic and hydrophilic residues on opposite sides of a cationic α-helix in AMPs have effective antibacterial properties [23,24]. Based on the amino acid combination, length, charge, and hydrophobicity of synthetic AMPs, they can be specifically designed to interact with specific regions of the microbial cell membrane [25,26]. Tryptophan (Trp, W) strongly interacts with zwitterionic liposomes that resemble cell membranes in a hydrophobic manner, and the highly hydrophobic aromatic ring of Trp is preferentially buried in the hydrophobic portion of the lipid bilayer. Additionally, negatively charged phosphate groups in microbial membranes prefer the polar regions of positively charged lysine (Lys, K) and arginine (Arg, R) residues. However, several AMPs are cytotoxic at higher doses and unsuitable for use in vivo, thus numerous studies have investigated novel peptides with antibacterial activity that do not display cytotoxicity at high concentrations [26,27].
In this study, we designed and synthesized a set of α-helical peptide analogs by substituting amino acids, particularly tryptophan, to compare the effects of differences in peptide length and amino acid position on antibacterial activity. The antibacterial efficacy of the synthesized peptides was evaluated against various drug-susceptible and drugresistant bacterial strains. The hemolytic and cytotoxic effects of the peptides on rat red blood cells (rRBCs) and HaCaT (human keratinocyte) cells were investigated. Finally, the mechanism of action of the peptide variants was examined in several liposomes, that acted as artificial cell membranes. Our findings provide evidence that the designed peptides can be used as antimicrobial agents for several natural infections without cytotoxic effects.

Designing and Modeling of α-Helical Peptides
Antimicrobial peptides (AMPs) act via intracellular and cell membrane modes of actions. The intracellularly mediated microbial killing and growth inhibition are due to flocculation of intracellular contents [28], alteration of cytoplasmic membrane septum formation [29], inhibition of cell wall [30], nucleic acid [31] and protein synthesis [32], and inhibition of enzymatic activity [33]. Unlike the mechanism of action of conventional antibiotics, the membrane-permeabilizing actions of AMPs, such as the 'toroidal pore model' [34,35], 'barrel-stave model' [34,35], and 'carpet model' [34,36], are considered as ways to overcome drug-resistance. Moreover, the selectivity of AMPs for bacterial membrane permeabilization, compared to host-cell membrane permeabilization, depends on cell membrane components. Therefore, the peptides used in this study were designed to facilitate the attachment and transmembrane action specifically on bacterial membranes. Previous studies on the adaptation of membrane proteins to the membrane-water interface provide useful information for designing membrane-acting peptides [37][38][39][40]. The interaction of amino acid residues with the membrane-water interface can be important for the binding, translocation, and function of AMPs. Thus, we used lysine (Lys), arginine (Arg), tryptophan (Trp), isoleucine (Ile), and glutamic acid (Glu) in our model peptides. Since the bacterial membrane is negatively charged, positively charged amino acids in the AMP sequence are required for attachment to the bacterial surface. Lys and Arg enable binding to the bacterial surface via electrostatic interactions, and their long and flexible alkyl chains are widely located in the hydrophobic core of the membrane. Glu was used to change the cation strength and induce an electrostatic force between peptides. In contrast, the Ile and Trp residues were used to complete the amphiphilic helical structure. In particular, the Trp side chain, with its indole NH, is close to the lipid carbonyl ester and is anchored rather rigidly to the interface. Therefore, the position of tryptophan in the peptide sequence may cause a change in the lipid orientation. Thus, we designed antimicrobial peptide analogs considering three issues: (1) the positions of Trp, (2) changes in cation strength by Lys, Arg, or Glu, and (3) the length of the α-helical structure. These peptides were designed by positional alterations of two tryptophan residues, substitution between lysine and arginine residues, net charges, and peptide lengths (10-mer and 14-mer).
The sequences of the 10 designed peptides are listed in Table 1. All peptides were expected to form a helical structure in partial or full sequence by using PEP_FOLD server ( Figure 1). Positioning the two tryptophan residues in the middle of the peptide sequence partially unraveled or loosened the helical structure ( Figure 1). The number of helical turns increased with sequence length; however, the WIKE-14 peptide in which Glu was substituted with Lys was predicted to form a helix-to-helix structure. expected to form a helical structure in partial or full sequence by using PEP_FOLD server ( Figure 1). Positioning the two tryptophan residues in the middle of the peptide sequence partially unraveled or loosened the helical structure ( Figure 1). The number of helical turns increased with sequence length; however, the WIKE-14 peptide in which Glu was substituted with Lys was predicted to form a helix-to-helix structure.

Cell-Selective Effects of Designed Peptides against Bacterial Strains and Mammalian Cells
The synthesized peptides were confirmed by RP-HPLC using C18 columns, and the molecular mass of each peptide was determined by MALDI-TOF/MS. We then investigated the antibacterial activities of the ten peptide variants against five pathogenic bacteria and compared them with those of melittin [41], an established antimicrobial agent (Table 1). Among the peptides with 10 amino acids, the MIC values of peptides with Arg residues were lower than those of peptides with Lys residues. Peptides with 14 amino acids showed better antibacterial activity than peptides with 10 amino acids. Among all peptides, those with Trp at the N-terminus (WIK-10, WIR-10, and WIK-14) had more potent antibacterial effects than the others. WIKE-14 had a higher MIC value than the other peptides. This indicates that the peptide binding affinity to the bacterial membrane is weakened due to lower cationic charge.
The antibacterial activity of the peptides was tested against 10 drug-resistant P. aeruginosa (DRPa) and S. aureus (DRSa) strains that were clinically isolated from patients and compared to conventional antibiotics (Figure 2A,B). Notably, three 14-mer peptides, KIW-14, KWI-14, and WIK-14, had increased potency against drug-resistant bacteria than six 10-mer peptide variants. In contrast, the MICs of all peptides were remarkably lower than those of the conventional antibiotics. In addition, peptides with Trp residues in the N-terminal region were more active than the others against drug-susceptible and drug-resistant bacteria.
The synthesized peptides were confirmed by RP-HPLC using C18 columns, and the molecular mass of each peptide was determined by MALDI-TOF/MS. We then investigated the antibacterial activities of the ten peptide variants against five pathogenic bacteria and compared them with those of melittin [41], an established antimicrobial agent (Table 1). Among the peptides with 10 amino acids, the MIC values of peptides with Arg residues were lower than those of peptides with Lys residues. Peptides with 14 amino acids showed better antibacterial activity than peptides with 10 amino acids. Among all peptides, those with Trp at the N-terminus (WIK-10, WIR-10, and WIK-14) had more potent antibacterial effects than the others. WIKE-14 had a higher MIC value than the other peptides. This indicates that the peptide binding affinity to the bacterial membrane is weakened due to lower cationic charge.
The antibacterial activity of the peptides was tested against 10 drug-resistant P. aeruginosa (DRPa) and S. aureus (DRSa) strains that were clinically isolated from patients and compared to conventional antibiotics (Figure 2A,B). Notably, three 14-mer peptides, KIW-14, KWI-14, and WIK-14, had increased potency against drug-resistant bacteria than six 10-mer peptide variants. In contrast, the MICs of all peptides were remarkably lower than those of the conventional antibiotics. In addition, peptides with Trp residues in the Nterminal region were more active than the others against drug-susceptible and drug-resistant bacteria.  AMPs must be nontoxic to be developed as effective antimicrobial therapeutics. Therefore, we evaluated the hemolytic and cytotoxic effects of all synthetic peptide variants on rat red blood cells (RBCs) and HaCaT cells. As shown in Figure 2C, melittin had a strong hemolytic effect even at a low concentration of 6.25 µM, whereas all 10 peptides showed low hemolysis even at a high concentration of 200 µM. On the other hand, in HaCaT cells, 200 µM melittin resulted in less than 20% cell survival, but the cells treated with the same concentration of the peptides survived, except for WIKE-14 ( Figure 2D). These findings suggest that the peptides exhibit selectivity between bacterial and human cells. To investigate the relationship between the antibacterial activity and permeability of the peptides, an inhibition zone assay was performed ( Figure 3). After adding 10 µL of the peptide solutions at the indicated concentrations to the punched wells, clear zones corresponding to the inhibitory effect of each peptide against E. coli and S. aureus were determined. As shown in Figure 3, KIW-14, KWI-14, and WIK-14 had increased inhibitory activity than other peptides and the size of the clear zone increased in a peptide dosedependent manner. These results were almost similar to the patterns of antimicrobial activity in the broth medium. Antimicrobial tests on agar plates can be used to understand the effect of viscosity on peptide activity and to compare the diffusion capacity of peptides. We propose that 14-mer peptides can rapidly inhibit the growth of surrounding bacteria due to their excellent diffusion ability. We expect that this ability can be applied in clinics for gel-type antibacterial pads or wound-healing bands. AMPs must be nontoxic to be developed as effective antimicrobial therapeutics. Therefore, we evaluated the hemolytic and cytotoxic effects of all synthetic peptide variants on rat red blood cells (RBCs) and HaCaT cells. As shown in Figure 2C, melittin had a strong hemolytic effect even at a low concentration of 6.25 µM, whereas all 10 peptides showed low hemolysis even at a high concentration of 200 µM. On the other hand, in HaCaT cells, 200 µM melittin resulted in less than 20% cell survival, but the cells treated with the same concentration of the peptides survived, except for WIKE-14 ( Figure 2D). These findings suggest that the peptides exhibit selectivity between bacterial and human cells.
To investigate the relationship between the antibacterial activity and permeability of the peptides, an inhibition zone assay was performed ( Figure 3). After adding 10 µL of the peptide solutions at the indicated concentrations to the punched wells, clear zones corresponding to the inhibitory effect of each peptide against E. coli and S. aureus were determined. As shown in Figure 3, KIW-14, KWI-14, and WIK-14 had increased inhibitory activity than other peptides and the size of the clear zone increased in a peptide dosedependent manner. These results were almost similar to the patterns of antimicrobial activity in the broth medium. Antimicrobial tests on agar plates can be used to understand the effect of viscosity on peptide activity and to compare the diffusion capacity of peptides. We propose that 14-mer peptides can rapidly inhibit the growth of surrounding bacteria due to their excellent diffusion ability. We expect that this ability can be applied in clinics for gel-type antibacterial pads or wound-healing bands.

Mode of Action of Peptides
To investigate the cellular distribution of the peptides in bacterial cells, E. coli cells were incubated with Flamma675-labeled peptides (BioActs, Incheon, Korea), and fluorescence was observed using confocal laser scanning microscopy (CLSM). As shown in Figure 4, all Flamma675-labeled peptides accumulated on E. coli surfaces, suggesting that the growth-inhibitory effect of the synthesized peptides against bacterial cells may result from interactions between the peptide analogs and the bacterial membrane.
Furthermore, to determine whether the peptides affected the permeability of the bacterial membrane, we performed a SYTOX Green uptake assay. SYTOX Green is an impermeable probe that enters the cell when the integrity of the cell membrane is changed or disrupted, resulting in binding to nucleic acids and emitting green fluorescence. Peptides at 1, 2, 4, and 8 µM concentrations were used to treat E. coli stabilized by pretreatment with SYTOX Green. Analysis using flow cytometry revealed that all peptides permeabilized the E. coli cell membrane and allowed the probe to penetrate, even at the lowest concentration of 1 µM ( Figure 5).

Mode of Action of Peptides
To investigate the cellular distribution of the peptides in bacterial cells, E. coli cells were incubated with Flamma675-labeled peptides (BioActs, Incheon, Korea), and fluorescence was observed using confocal laser scanning microscopy (CLSM). As shown in Figure 4, all Flamma675-labeled peptides accumulated on E. coli surfaces, suggesting that the growth-inhibitory effect of the synthesized peptides against bacterial cells may result from interactions between the peptide analogs and the bacterial membrane.   Furthermore, to determine whether the peptides affected the permeability of the bacterial membrane, we performed a SYTOX Green uptake assay. SYTOX Green is an impermeable probe that enters the cell when the integrity of the cell membrane is changed or disrupted, resulting in binding to nucleic acids and emitting green fluorescence. Peptides at 1, 2, 4, and 8 µM concentrations were used to treat E. coli stabilized by pretreatment with SYTOX Green. Analysis using flow cytometry revealed that all peptides permeabilized the E. coli cell membrane and allowed the probe to penetrate, even at the lowest concentration of 1 µM ( Figure 5).   To further investigate the bacterial growth inhibitory effects of the synthesized peptides, we used the membrane potential-sensitive fluorescent probe DiSC3-5 to assess the ability of the peptides to induce membrane depolarization in antibiotic-susceptible E. coli (ATCC 25922). The peptide-induced changes in membrane permeability leading to dissipation of the transmembrane potential were monitored by measuring the increase in fluorescent emission caused by the release of the membrane potential-sensitive dye, DiSC3-5. As shown in Figure 6A, the magnitude of the bacterial membrane depolarization by the peptides and melittin varied significantly, whereas the maximum depolarization of E. coli treated with 10-mer peptides was detected at a peptide concentration of 16 µM. The depolarization of 14-mer peptide treated cells, except WIKE-14, was detected at about 4 µM, the MIC for E. coli strains. The dose-dependent dissipation of the bacterial membrane potential by the peptides indicated a correlation between the antibacterial activity and membrane potential disruption. These results suggest that 14 amino acid peptides were more potent in inducing membrane depolarization in intact E. coli than the 10 amino acid peptide analogs.
To further investigate the bacterial growth inhibitory effects of the synthesized peptides, we used the membrane potential-sensitive fluorescent probe DiSC3-5 to assess the ability of the peptides to induce membrane depolarization in antibiotic-susceptible E. coli (ATCC 25922). The peptide-induced changes in membrane permeability leading to dissipation of the transmembrane potential were monitored by measuring the increase in fluorescent emission caused by the release of the membrane potential-sensitive dye, DiSC3-5. As shown in Figure 6A, the magnitude of the bacterial membrane depolarization by the peptides and melittin varied significantly, whereas the maximum depolarization of E. coli treated with 10-mer peptides was detected at a peptide concentration of 16 µM. The depolarization of 14-mer peptide treated cells, except WIKE-14, was detected at about 4 µM, the MIC for E. coli strains. The dose-dependent dissipation of the bacterial membrane potential by the peptides indicated a correlation between the antibacterial activity and membrane potential disruption. These results suggest that 14 amino acid peptides were more potent in inducing membrane depolarization in intact E. coli than the 10 amino acid peptide analogs.  Table 2). The use of two buffers is to understand the antimicrobial activity in low ionic strength (buffer I, topical treatments) and high ionic strength (buffer II, physiological environment of human fluids) conditions. In aqueous conditions without any liposomes, the maximum emission intensity of all antibacterial peptides was observed at 352-357 nm in buffer I and II. However, in the presence of PE:PG vesicles (bacterial membrane-mimicking environments), the maximum Trp emission intensity was detected at 318-343 nm in buffer I, suggesting that all antibacterial peptides strongly interacted with the bacterial membrane, and the interaction shifted the Trp intensity of the peptides. The maximum emission intensity of all antibacterial peptides at 318-346 nm in buffer II reflects their binding to the bacterial membrane. In the presence of PG:CL (1:1, w/w) vesicles (inner membrane mimicking environments of Gram-negative bacteria), the results were similar to those in the PE:PG condition. On the other hand, in PC:CH:SM (1:1:1, w/w/w) liposomes (mammalian membrane-mimicking environments), the maximum Trp emission of the designed peptides shifted to 0-6 nm compared with the aqueous condition, indicating no interaction with PC:CH:SM liposomes. These results were consistent with the lack of cytotoxic effects of the synthetic peptides ( Figure 2C,D). To further investigate the effect of the peptides on cell membranes, the membranepermeabilizing abilities of the designed peptides were measured by calcein leakage from various cell membrane-mimicking liposomes, which comprised PE:PG (7:3, w/w) as the bacterial membrane and PC:CH:SM (1:1:1, w/w/w) as the mammalian plasma membrane analogs. The dye leakage value indicates the ability of an antimicrobial agent to disrupt the membranes. As shown in Figure 6B, bacterial liposomes showed a significant dosedependent release of calcein in the presence of melittin or 14-mer peptides, such as KIW-14, KWI-14, WIK-14, and WIKE-14. The 10-mer peptides either showed no significant release of calcein (KIW-10, KWI-10, and WIK-10) or a weak leakage (RIW-10, RWI-10, and WIR-10) ( Figure 6B). These results suggest that the 14-mer peptides directly disrupt the bacterial membrane, and the 10-mer peptides only weakly damage the membrane during bacterial membrane penetration. However, none of the peptides exhibited remarkable calcein release from the mammalian plasma membrane, whereas melittin elicited significant membrane disruption even at low concentrations ( Figure 6B). These results are consistent with the observed antibacterial and cytotoxic effects.
Morphological changes on the surface of E. coli cells incubated with the indicated peptides were observed using scanning electron microscopy (SEM). The peptide-untreated cells exhibited a smooth surface without cell debris or any changes (Figure 7, control). However, the peptide-treated cells were wounded and shrunken with small blebs, indicating damage to cell membranes by the peptides (Figure 7). Thus, our findings suggest that the designed peptides exhibit potent antibacterial activity by destabilizing the cell membrane or by membranolysis.
Morphological changes on the surface of E. coli cells incubated with the indicated peptides were observed using scanning electron microscopy (SEM). The peptide-untreated cells exhibited a smooth surface without cell debris or any changes (Figure 7, control). However, the peptide-treated cells were wounded and shrunken with small blebs, indicating damage to cell membranes by the peptides (Figure 7). Thus, our findings suggest that the designed peptides exhibit potent antibacterial activity by destabilizing the cell membrane or by membranolysis.

In Vivo Antibacterial Effects of the Selected Peptides
Pseudomonas aeruginosa is the most commonly isolated microorganism in patients with nosocomial pneumonia. To elucidate the in vivo antibacterial effects of WIK-10, WIK-14, and WIKE-14 peptides against drug-resistant P. aeruginosa infection, we administered high concentrations of the bacteria by intratracheal (i.t.) intubation in an acute pneumonitis model. Peptides were administered i.t. to enable rapid action without interference from proteolytic enzymes. After bacterial infection, all mice in the PBS-treated group died within 3 days. Although the mice in the peptide-treated group survived for a long time, the endpoint was set at 3 days. To analyze the severity and progression of chronic pulmonary disease, Alcian blue staining, H&E staining, and immunostaining were performed using paraffin sections from the excised tissue. Alcian blue is used to visualize acidic epithelial and connective tissue mucins. As shown in Figure 8, bacteria-infected tissues had apparent mucus deposition, which was reduced in tissues with peptide treatment, similar to uninfected tissues. H&E staining (Figure 8

In Vivo Antibacterial Effects of the Selected Peptides
Pseudomonas aeruginosa is the most commonly isolated microorganism in patients with nosocomial pneumonia. To elucidate the in vivo antibacterial effects of WIK-10, WIK-14, and WIKE-14 peptides against drug-resistant P. aeruginosa infection, we administered high concentrations of the bacteria by intratracheal (i.t.) intubation in an acute pneumonitis model. Peptides were administered i.t. to enable rapid action without interference from proteolytic enzymes. After bacterial infection, all mice in the PBS-treated group died within 3 days. Although the mice in the peptide-treated group survived for a long time, the endpoint was set at 3 days. To analyze the severity and progression of chronic pulmonary disease, Alcian blue staining, H&E staining, and immunostaining were performed using paraffin sections from the excised tissue. Alcian blue is used to visualize acidic epithelial and connective tissue mucins. As shown in Figure 8, bacteria-infected tissues had apparent mucus deposition, which was reduced in tissues with peptide treatment, similar to uninfected tissues. H&E staining (Figure 8  To investigate the expression of the proinflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor α (TNF-α), PE-labeled cytokine antibodies were used in each tissue. Mice treated with the peptides had greater inhibition of cytokine secretion than those treated with PBS. Interestingly, mice treated with WIKE-14 showed slightly more inflammatory cell infiltrates than those treated with WIK-14, but secreted fewer cytokines.
Lipopolysaccharides in the outer membrane of Gram-negative bacteria and teichoic acid in the peptidoglycan layer of Gram-positive bacteria contain phosphate, indicating that all bacteria have a negatively charged surface. Positively charged amino acids, such as lysine and arginine, in AMPs facilitate attractive binding to bacteria via electrostatic interactions. Membrane-acting AMPs must pass through the outer membrane or the peptidoglycan layer into the cytoplasmic membrane, where they exhibit membranolytic action. Figure 9 shows the expected actions of the peptides on the cytoplasmic membrane. The amphipathic helical structure is induced by the attachment of Lys or Arg to the membrane surface. Because lysine and arginine have long aliphatic parts, they tend to be located in the hydrophobic part of the bilayer. Although the Trp residues in our designed peptides were hydrophobic, the amide group of the fused aromatic ring was preferentially located in a polar environment. However, since Ile has an aliphatic chain, it has the property of making hydrophobic bonds with lipid fatty acids, resulting in insertion into the membrane. All peptides act similarly on the cytoplasmic membrane, but the range of their action on the membrane is widened based on the position of the Trp residues and the distance between Trp residues. We propose that the superior antibacterial activity of WIK-10, WIR-10, and WIK-14 is due to the long distance between the Trp residues, which  .) administration, Balb/c mice that were treated with PBS and the indicated peptides (n = 4) by i.t. administration were euthanized after three days. Inflammatory effects of the peptides were examined by immunohistology using lung tissues of P. aeruginosa-induced pneumonia mice. TNF-α and IL-6 were detected using monoclonal PE-label antibody.
To investigate the expression of the proinflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor α (TNF-α), PE-labeled cytokine antibodies were used in each tissue. Mice treated with the peptides had greater inhibition of cytokine secretion than those treated with PBS. Interestingly, mice treated with WIKE-14 showed slightly more inflammatory cell infiltrates than those treated with WIK-14, but secreted fewer cytokines.
Lipopolysaccharides in the outer membrane of Gram-negative bacteria and teichoic acid in the peptidoglycan layer of Gram-positive bacteria contain phosphate, indicating that all bacteria have a negatively charged surface. Positively charged amino acids, such as lysine and arginine, in AMPs facilitate attractive binding to bacteria via electrostatic interactions. Membrane-acting AMPs must pass through the outer membrane or the peptidoglycan layer into the cytoplasmic membrane, where they exhibit membranolytic action. Figure 9 shows the expected actions of the peptides on the cytoplasmic membrane. The amphipathic helical structure is induced by the attachment of Lys or Arg to the membrane surface. Because lysine and arginine have long aliphatic parts, they tend to be located in the hydrophobic part of the bilayer. Although the Trp residues in our designed peptides were hydrophobic, the amide group of the fused aromatic ring was preferentially located in a polar environment. However, since Ile has an aliphatic chain, it has the property of making hydrophobic bonds with lipid fatty acids, resulting in insertion into the membrane. All peptides act similarly on the cytoplasmic membrane, but the range of their action on the membrane is widened based on the position of the Trp residues and the distance between Trp residues. We propose that the superior antibacterial activity of WIK-10, WIR-10, and WIK-14 is due to the long distance between the Trp residues, which causes significant cell membrane damage.

Solid-Phase Peptide Synthesis by Microwave
Peptides were synthesized by a Liberty Microwave Peptide synthesizer (CEM Co.). To generate amidated peptides at carboxyl-termini, Rink Amide ProTide Resin (0.58 mmol/g) was used. Fmoc deprotection was completed using 20% piperidine in N,N-Dimethylformamide (DMF). Coupling of each Fmoc-amino acid was achieved in the presence of diisopropyl carbodiimide and Oxyma in DMF. To generate N-terminal fluorescently labeled peptides, Flamma675-carboxylic acid was added to the peptide-bound resin. The protecting groups and peptides on the resin were removed using trifluoroacetic acid(TFA)/diH2O/triisopropylsilane (95: 2.5: 2.5, v/v/v) for 40 min at 40 °C. The filtrated peptide solution was precipitated and washed in ice-cold diethyl ether. The solid powder was isolated by centrifugation and then dried under a vacuum. The purification of crude

Solid-Phase Peptide Synthesis by Microwave
Peptides were synthesized by a Liberty Microwave Peptide synthesizer (CEM Co.). To generate amidated peptides at carboxyl-termini, Rink Amide ProTide Resin (0.58 mmol/g) was used. Fmoc deprotection was completed using 20% piperidine in N,N-Dimethylformamide (DMF). Coupling of each Fmoc-amino acid was achieved in the presence of diisopropyl carbodiimide and Oxyma in DMF. To generate N-terminal fluorescently labeled peptides, Flamma675carboxylic acid was added to the peptide-bound resin. The protecting groups and peptides on the resin were removed using trifluoroacetic acid(TFA)/diH 2 O/triisopropylsilane (95: 2.5: 2.5, v/v/v) for 40 min at 40 • C. The filtrated peptide solution was precipitated and washed in ice-cold diethyl ether. The solid powder was isolated by centrifugation and then dried under a vacuum. The purification of crude peptides was done on a C18 column (Zorbax, 21.2 × 250 mm, 300Å, 7-µm) on a Shimadzu semi-preparative HPLC system, using a 10-90% acetonitrile gradient in water with 0.05% TFA for 80 min. The purity and molecular masses of the isolated peptide were measured on an analytical HPLC system and a matrix-assisted laser desorption ionization (MALDI) mass spectrometer (Kratos Analytical Ins.), respectively.

Antibacterial Assay by a Microdilution Method
Pseudomonas aeruginosa (ATCC 15692), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 25923) were purchased from the American Type Culture Collection. Listeria monocytogens (KCTC 3710) and Bacillus subtilis (KCTC 1998) were from the Korean Collection for Type Cultures. Drug-resistant S. aureus (DRSa-001~005) and P. aeruginosa (DRPa-001~005) were collected from Paik Hospital of Inje University in Busan, South Korea. Pre-cultured bacteria grown to mid-log phase were suspended in 10 mM sodium phosphate (SP, pH 7.2) or phosphate-buffered saline (PBS, pH 7.2) with 10% culture media. Bacterial suspensions were added to each peptide dilute (final 5 × 10 5 colony-forming units (cfu)/mL), after which the plates were incubated at 37 • C for 24 h. The turbidity of each well was measured at the absorbance at 600 nm and the morphology of bacterial growth was observed under light microscopy. MICs (Minimum inhibitory concentrations) were defined as the lowest concentration of peptide that expresses the complete inhibition of bacterial growth [42,43].

Hemolysis and Cytotoxicity
Fresh rat erythrocytes were collected and washed by centrifugation at 1000× g for 10 min with PBS. The rat RBCs (8% (v/v)) were added in a two-fold serially diluted peptide with PBS, followed by incubation for 1 h at 37 • C. After centrifugation of samples at 1000× g for 10 min, the supernatants were collected and their absorbance was measured at 414 nm. The 8% RBCs dissolved in PBS and RBCs treated with Triton X-100 were considered as the negative and positive controls, respectively. The hemolysis activity was calculated using the formula, % hemolysis = [(Abssample − Absnegative)/(Abspositive − Absnegative)] × 100 [43,44].
To examine the cytotoxic effect of peptides, HaCaT (human keratinocyte) cells were sub-cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 U/mL penicillin at 37 • C in a 5% CO 2 chamber. The cells seeded into 4 × 10 3 cells/well were incubated for 24 h. Peptide solutions diluted with DMEM were incubated with cells for 24 h at 37 • C. 3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyltetrazolium Bromide (MTT) solution was added to the cells, followed by additional incubation for 4 h. After removing the media, 200 µL of dimethyl sulfoxide was added to the wells. Absorbance of each well was measured at 570 nm using a microtiter reader (Molecular Devices Emax, CA, USA) [44,45].

Inhibition Zone Assay
E. coli and S. aureus cells were washed with SP buffer. Fifteen milliliters of underlay agarose gel containing 0.03% (w/v) culture medium, and 1% (w/v) agarose in the presence of 5 × 10 5 cfu/mL, was poured into a Petri dish, and the gel was solidified. Peptide solution (8 µL) was added to the punched wells (3 mm diameter) after which the plate was incubated at 37 • C for 3 h. A gel solution containing 6% culture media and 1% agarose was poured on underlay gel, followed by further incubation at 37 • C for 24 h.

Membrane Depolarization
The membrane-depolarizing activity of peptides was determined using DiSC3-5, a membrane potential-sensitive dye, on intact S. aureus and E. coli cells. Pre-grown bacteria were washed three times with 5 mM HEPES buffer containing 20 mM glucose (pH 7.2). The cells were suspended to A 600 = 0.05 in 5 mM HEPES buffer containing 20 mM glucose and 0.1 M KCl (pH 7.2), and the dye was added to a final concentration of 100 nM and then incubated for about 60 min until a constant fluorescence was achieved. Peptides were then added to bacterial suspensions. The fluorescence was recorded (Ex 622 nm and Em 670 nm) for 30 min [43].

Confocal Laser-Scanning Microscopy (CLSM)
E. coli treated with Flamma ® 675-labeled peptides were observed on a CLSM. Cells were inoculated and adjusted as the above procedure of antimicrobial assay. Flamma675labeled peptides were added to 100 µL of the cell suspensions at each MIC, after which the cells incubated for 30 min were washed by centrifugation at 4000 rpm for 5 min in three times with ice-cold PBS buffer. Localization of Flamma675-labeled peptides was then examined using CLSM (A1R HD 25; Nikon, Tokyo, Japan).

SYTOX Green Uptake Assay
Pre-grown E. coli cells were washed and suspended to 2 × 10 7 cells/ ml in SP buffer. The cells pre-treated with 1 µM SYTOX green for 15 min in the dark were incubated with the peptides in appropriate concentrations; the bacteria were analyzed by flow cytometry (Attune NxT acoustic focusing cytometer, Thermo Fisher Scientific Co., Waltham, MA, USA) [44].

Small Unilamellar Vesicles Preparation
SUVs were prepared by sonication and the freeze-thaw method, respectively [27,28]. The lipid mixtures in chloroform were dried in a glass tube under nitrogen, after which they were lyophilized overnight. Dry lipid films suspended in 2 mL buffer preheated at 50 • C were vortexed. SUVs were prepared by sonication in a bath-type sonicator for 30 min. After the vesicles were prepared, unilamella vesicles were extruded 15 times through 0.05 µm polycarbonate membranes for SUVs using an Avanti Mini-Extruder (Avanti Polar Lipids inc., Alabaster, AL, USA), to ensure a homogeneous population. Their concentration was determined by a standard phosphate assay [44].

Tryptophan Fluorescence Blue Shift
To analyze peptide binding and partitioning on/into the lipid bilayer, the fluorescence emission spectrum of Trp residues was monitored upon the addition of SUVs, composed of PC/CH/SM (1:1:1, w/w/w) or PE/PG (7:3, w/w). A 2 µM of peptide solution was added to 1 mL of buffers or vesicles at a total lipid concentration range of 0 to 600 µM. This assay was performed in two buffers: buffer I, 10 mM HEPES containing 10 mM NaCl (pH7.4) and buffer II, 10 mM HEPES containing 150 mM NaCl and 5 mM MgCl 2 (pH 7.4). Tryptophan fluorescence was excited at 280 nm, and the emission spectrum was measured from 290 to 510 nm in 1 nm increments with 1 sec signal averaging. Background and lipid scattering signals were subtracted. The wavelength of the maximum fluorescence emission was recorded [44].

Scanning Electron Microscopy (SEM) Preparation
Pre-cultivated E. coli cells (1 × 10 8 cells/mL) were incubated with peptides at a MIC value for 2 h. Bacterial cells were attached on glass slides coated by poly-L-Lysine coated and fixed with 100 mM HEPES buffer containing 2% glutaraldehyde (v/v) (pH 7.4) for 12 h at RT. The PBS-washed cells were subsequently dehydrated in an OTTIX shaper (Diapath S.p.A, Bergamo, Italy), followed by chemical dry with hexamethyldisilazane. Samples were sputter-coated by gold-palladium and observed under field emission SEM (JSM-7100F; JEOL, Ltd., Tokyo, Japan).

Murine Infection Model
All animal experiments and procedures were investigated with the approval of the institutional animal care and use committee (IACUC) in Sunchon national university, South Korea (SCNU IACUC-2019-10). Ten-week-old female Balb/c mice (Koatech Co., Pyongtaek, Gyeonggido, Korea) were anesthetized by inhalation of isoflurane in pure oxygen and instilled intratracheally with 1 × 10 8 cfu/mL of DRPA-005 in 100 µL of PBS. After one hour of exposure, each peptide was intratracheally administered at the indicated dose in 50 µL of PBS. Mice were monitored for signs of morbidity and euthanized on the third day after bacterial exposure. Mice were euthanized by CO 2 inhalation, and the infected lung tissues were excised and fixed in 4% paraformaldehyde. The fixed tissues were dehydrated using a series of ethanol solutions (50−100%) and embedded in paraffin. The tissues were sectioned in a thickness of 5 µm (Leica microtome, Deerfield, IL, USA). Histopathology of the lung tissues was performed by Alcian blue and hematoxylin and eosin (H&E) staining. Immunohistology was analyzed by using the monoclonal antibody of PE-conjugated anti-IL-6 and anti-TNF-α (BioLegend, San Diego, CA, USA). The stained tissue sections were observed under a fluorescence microscope (OPTINIT KCS3-160S; Korea Lab Tech).

Statistical Analysis
The mean values of at least four independent determinations ± SD were calculated using Excel software (Student t-test).

Conclusions
In summary, we designed 10 peptide analogs with repositioned Trp based on Arg, Glu, Lys, Ile, and Trp amino acids. Considering the balance between antibacterial activity and cytotoxicity, 10-mer peptides using Lys have less antibacterial activity and cytotoxicity than Arg-containing peptides, and 14-mer peptides showed greater antibacterial activity and cytotoxicity than 10-mer peptides. When Trp was located near the N-terminus, antibacterial activity and toxicity simultaneously increased at a high concentration (200 µM). However, their therapeutic index is higher than that of peptides with other Trp-positioning, indicating that they have better potential for clinical applications. Interestingly, WIKE-14, with reduced cation strength, had lower antibacterial activity than other peptides in vitro, but had increased antibacterial activity in vivo. In particular, the peptide inhibitory effects on cytokine secretion showed the strongest effects among the tested parameters. All peptides exerted antibacterial activity by destabilizing the cell membrane or by membranolysis. Although further research is required, our findings indicate that at least three helix turns are needed for the clinical application of membrane-active AMPs.


s2fieldsofstudy: Biology Medicine

text:
Chronic Inflammatory Demyelinating Polyneuropathy Post-mRNA-1273 Vaccination

Massive efforts are being made to develop coronavirus disease 2019 (COVID-19) vaccines at an unprecedented rate. The vaccinations' adverse impact profile, on the other hand, has not been well established. Neurological complications are increasingly reported as a result of these vaccines. One such complication identified is immune-mediated inflammatory polyneuropathy, which affects peripheral nerves and neurons. We report a case of chronic inflammatory demyelinating polyneuropathy (CIDP) post-mRNA-1273 (Moderna) COVID-19 vaccine. Recognizing this complication and distinguishing it from Guillain-Barré syndrome enables timely initiation of treatment. Additionally, our report highlights a possible link between vaccination and subsequent development of CIDP, but conclusive evidence of a causal relationship requires more extensive studies.

Introduction
The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a global pandemic in March 2020. COVID-19 viral infection is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. COVID-19 vaccines are being developed at a rapid pace. However, the vaccine's side effect profile is still undetermined. Complications involving the nervous system are increasingly being reported due to the result of these vaccines. One such adverse event reported is immune-mediated inflammatory polyneuropathy. It is further classified as acute or chronic depending on the presentation and duration of symptoms.
Viruses and vaccines can induce autoimmune neuropathies via various mechanisms, including antigen mimicry, cytokine overexpression, and activation of self-reactive T-cell clones, resulting in aberrant major histocompatibility complex (MHC) class II expression [2]. The linkage of the swine flu vaccine with Guillain-Barré syndrome (GBS) in 1976 highlighted the possibility of inflammatory neuropathy related to vaccination [3]. Subsequently, research revealed that the collective GBS risk from influenza was considerably higher than that associated with influenza vaccinations [4].
Numerous vaccines have been associated with various neurological problems, including seizures with the pertussis vaccine, meningoencephalitis with the Japanese encephalitis vaccine, and giant cell arteritis with the influenza vaccine. Transverse myelitis, encephalitis, GBS, and optic neuritis have been documented following vaccination with human papillomavirus, rubella, yellow fever, measles, tetanus, rabies, and hepatitis A or B [5]. COVID-19 vaccines are not exempt from such adverse effects. Due to the severity of the COVID-19 pandemic, massive efforts were made to shorten the timing of vaccine development. Neurological problems associated with SARS-CoV-2 vaccinations are rare [6]. In their comprehensive analysis, Chen et al. described various neurological adverse effects associated with routinely available COVID-19 vaccinations [7,8]. Most of the neurological symptoms reported following vaccination are mild and transient, such as headache, dizziness, myalgia, and paresthesia. A few case reports have described more serious neurological symptoms such as GBS, transverse myelitis, and cranial nerve neuropathies [9][10][11]. Garg and Paliwal reviewed published studies on COVID-19 vaccines associated with neurological complications. The review detailed published reports of various neuroinflammatory, neurovascular, peripheral nerve, and neuromuscular complications [11]. Such severe neurological manifestations were also reported in the Vaccine Adverse Event Reporting System (VAERS) regarding all the COVID-19 vaccines [12,13]. Fernandes et al. documented a case series of four patients. Two of them developed new-onset seizures and transverse myelitis following immunization with the Pfizer-BioNTech vaccine. Other two developed meningitisretention syndrome and GBS following vaccination with the ChAdOx1 nCoV-19 (AstraZeneca) vaccine [14].
Outside VAERS of the CDC, the reported data on COVID-19 vaccination-related chronic inflammatory demyelinating polyneuropathy (CIDP) are scarce. As of March 2022, there are 22 reported events in VAERS in the USA when searched under CIDP and Moderna vaccine. Out of 22 reported events, one event is documented by a healthcare provider confirming CIDP, and one event is documented by ER provider suggestive of CIDP without further diagnostic confirmation. Eight events were documented by patients or family members without citing confirmation of diagnosis through a healthcare provider or any mention of administration of relevant therapeutics, but event descriptions were consistent with neurological symptomatology in line with CIDP. Three events reported by patients or family members cited the confirmation of diagnosis through a healthcare provider. Two events reported by patients were suggestive of a relapse of CIDP rather than a new occurrence. Seven events reported by patients were not consistent with CIDP or any neurological symptomatology. Therefore, probably wrongly reported under CIDP. There is no reported published report on CIDP associated with the Moderna vaccine.
We present a case of an elderly female who developed lower extremities weakness gradually over five months. Clinical, pathognomonic, lab, and electromyography (EMG) findings were consistent with CIDP. Outside a few reports in VAERS, this is the first case of CIDP documented following immunization with the Moderna COVID-19 (mRNA-1273) vaccine.

Case Presentation
The patient was a 66-year-old female with a past medical history significant for type 2 diabetes mellitus with neuropathy, hypertension, and hyperlipidemia, who presented to the hospital with a five-month history of progressive lower extremity weakness and difficulty walking, and frequent falls. Her A1C was normal, and she was treated with basal insulin, short-acting insulin, and metformin. Eight months prior to admission, the patient had received Moderna COVID-19 two vaccine series. She experienced mild fever and malaise after the second dose, followed by residual fatigue lasting several weeks. Three months after that, she woke up with no feeling in both her legs and right arm weakness. She was admitted to the hospital for a stroke workup. She was hospitalized for evaluation of a transient ischemic attack. Brain imaging was unremarkable. The stroke workup was negative. She was discharged on aspirin and atorvastatin with a neurology follow-up. She also had a urinary tract infection identified, which was treated with appropriate antibiotics.
Her leg weakness kept on gradually worsening over the next two months when she also started having electric, shooting pain moving upwards from toes to hips and both legs. She also started noticing more bilateral arm weakness. Further, over the course of the next three months, she had a faster progression of her symptoms. She started using a wheelchair, as she could not get up from the couch. Her frequency of falls gradually increased to one to two daily falls on average. Symptoms were now associated with numbness and tingling in bilateral upper extremities, poor oral intake, and unintentional weight loss of 40 pounds over eight months. Bilateral leg weakness followed a waxing and waning pattern with an overall gradual decline in her strength. She tried as-needed ibuprofen for her extremity pain. She was evaluated by neurology service and was found to have hyporeflexia on the exam. She had unremarkable cervical, thoracic, and lumbar spine magnetic resonance imaging (MRI). No abnormal spinal cord signal, intrathecal enhancement, or thecal sac stenosis was seen. She had an unremarkable screening colonoscopy.
She was subsequently hospitalized for further evaluation and underwent extensive workup. Physical examination revealed decreased motor strength in all extremities involving proximal and distal muscles, more in the legs than arms. Decreased sensations of light touch were noted in all extremities. She had absent deep tendon reflexes in both lower extremities. Muscle tone and bulk were normal. Her complete blood count, comprehensive metabolic panel, erythrocyte sedimentation rate, C-reactive protein, creatine kinase, and thyroid-stimulating hormone were unremarkable. Serum protein electrophoresis showed a questionable protein band that was suspicious for being a monoclonal protein in the gamma globulin region. Serum immunofixation revealed IgG kappa monoclonal protein and a slightly abnormal IgG lambda monoclonal protein, which was not apparent in the less sensitive serum electrophoresis study. Urine protein electrophoresis showed predominantly glomerular proteinuria with an additional protein band that could potentially be a monoclonal protein. Urine immunofixation revealed IgG kappa monoclonal protein. Vitamin B1, folate, vitamin D, copper, and vitamin B12 levels were within the normal range. Lumbar puncture revealed clear CSF and albuminocytological dissociation as demonstrated in Table 1. CSF protein was 237 mg/dL, glucose was 56 mg/dL, and total nucleated cell (TNC) count was 2. CSF angiotensin-converting enzyme was normal. Serology testing, including antinuclear antibody (ANA), anti-double-stranded DNA, HIV, and West Nile virus, was negative. CSF polymerase chain reaction (PCR) testing for West Nile virus was negative. Coronavirus SARS-CoV-2 PCR testing was negative. MRI of the brain was unremarkable.  Nerve conduction studies demonstrated an absent sural sensory nerve response. The peroneal motor nerve response distal latencies were prolonged with significantly reduced amplitudes and significantly slowed proximal conduction velocities. The tibial motor nerve response distal latencies were significantly prolonged with reduced amplitudes. The F response was absent on the right and prolonged on the left. The nerve conduction results were consistent with prolonged distal latencies and decreased amplitudes in the bilateral peroneal and tibial motor nerve conductions, absent right sural sensory potential, and widespread active and chronic denervation in all muscles tested in both lower extremities. This pattern supported the diagnosis of CIDP with both axonal and demyelinating features. Concentric needle electromyography of the lower extremities demonstrated diffusely abnormal insertional activity with both fibrillations and positive sharp waves in all the tested muscles. The activated motor units of the lower extremities demonstrated increased motor unit complexities, increased amplitudes, and reduced recruitment patterns. In conclusion, electrodiagnostic findings were consistent with severe bilateral neuropathy with acute and chronic denervation changes consistent with CIDP. There were no findings to suggest myopathy or myositis ( Tables  2-5).     She was treated with a 2 gm/kg dosing regimen of intravenous immunoglobulin (IVIG) therapy. She was discharged to a rehabilitation facility. At the three-month follow-up, significant improvement in her symptoms was noted. She was continued on IVIG treatment with a 0.6 gm/kg x two days every four weeks regimen.

Discussion
Due to the significant prevalence of neurological diseases in the population and the vast number of vaccinated people, some of these conditions will manifest within the post-vaccination timeframe. As a result, establishing a causal link between vaccination and neurological disease will be a tough endeavor.
The current vaccines in use in the USA are Moderna (mRNA-1273), Pfizer-BioNTech (BNT162b2), and Johnson & Johnson's Janssen (Ad26.COV2.S). These vaccines are generally well-tolerated. Mild symptoms such as pain at the injection site, redness, fevers, headaches, or myalgias are common side effects. Numerous neurological adverse effects have been recorded following the administration of all current COVID-19 vaccines, most notably after Ad26.COV2.S and ChAdOx1 nCoV-19 (Oxford/AstraZeneca). While the occurrence of GBS is recognized in COVID-19 patients, several GBS cases have been associated with COVID-19 vaccines, particularly those containing adenoviral components. Neuropathies following vaccination are uncommon, and CIDP development during the post-vaccination interval is exceedingly rare, occurring in less than 2% of the individuals [15]. Moreover, CIDP occurrence remains poorly characterized [16]. Kim [23]. The authors conducted an extensive search of databases. Interestingly, they did not discover any report linking COVID-19 to a new onset CIDP. Few reports mentioned the exacerbation of CIDP symptoms with COVID-19 infection in known CIDP patients. Notably, because the GBS patients in the study were not tracked prospectively, it is unknown whether some of them had acute-onset CIDP, which has been described in up to 5-16% of patients in pre-COVID-19 investigations [24]. The authors were able to note a similar pattern of GBS incidence in post-COVID-19 vaccination patients. Also, they did not notice exacerbation of CIDP post-COVID-19 vaccination in existing CIDP patients. There is convincing evidence regarding acute inflammatory demyelinating polyneuropathy (AIDP) following vaccination with COVID-19 and COVID-19 vaccines (including Moderna). Abo-Zed and Pinevich reported a case of GBS that developed shortly after the Moderna COVID-19 vaccine, which subsequently evolved into CIDP [25]. Of note, this patient had a history of GBS/AIDP four years ago after receiving the influenza vaccine. Whereas our case did not have any history of AIDP/GBS or CIDP. Souza et al. reported a case series of four patients with CIDP after the ChAdOx1 nCoV-19 (AstraZeneca) vaccine [26]. While there have been a few studies examining the relationship between immunizations and chronic inflammatory neuropathies, most of them had limitations such as recall bias, retrospective design, and difficulty in precisely establishing the onset of disorders.
GBS and CIDP are immune-mediated illnesses in which the precise mechanism behind the pathophysiology of the immune response is unknown. GBS pathogenic episodes can generate antibodies against peripheral nerves and myelin sheath epitopes, leading to an autoimmune process. CIDP is often an idiopathic condition in which an immune response is directed against myelin components due to an autoimmune process involving primarily immunological mechanisms mediated by T cells. Hence, cellular immune mechanisms are a vital feature of CIDP. Infiltration of macrophages into nerves and perivascular inflammation on nerve biopsies imply that cell-mediated mechanisms are involved in demyelination. CIDP patients may have elevated T helper cells in the CSF [27]. Segmental demyelination and remyelination are characteristics of CIDP and repeatedly occur over time, resulting in onion bulb development via Schwann cell process growth.
The distinction between GBS and CIDP is important. GBS is the most prevalent cause of acute flaccid paralysis, characterized by autonomic dysfunction, sensory abnormalities, and varying degrees of weakness.
Although the specific pathophysiology is unknown, this disorder is believed to result from an autoimmune response. GBS is often preceded by a respiratory or gastrointestinal infection. CIDP is a chronic disorder and the most common autoimmune polyneuropathy in adults. As per established criteria, a time period of more than eight weeks to recognize the greatest weakness in CIDP is used to differentiate it from AIDP, which reaches a nadir in four weeks or less. CIDP is characterized by a subacute to chronic onset that typically takes more than eight weeks to manifest paresthesias, loss of sensations, proximal and distal weakness, areflexia, asymmetric conduction velocity slowing with conduction-block like characteristics on electrodiagnostic testing, and association with albuminocytologic dissociation (elevated CSF protein without pleocytosis) [28]. Nerve pathology in CIDP shows segmental demyelination, onion bulb formation, perivascular inflammation, and axonal degeneration [28]. Patients often present with a relapsing and remitting course. Compared to AIDP, autonomic symptoms and back pain are less common in CIDP. In comparison to AIDP, the occurrence of respiratory failure leading to ventilator support or bulbar involvement is rare in CIDP.
CIDP is diagnosed based on specific clinical history and symptoms. CSF analysis, electrophysiological studies, and nerve biopsies are all important diagnostics, beneficial in excluding other possible diagnoses than in identifying CIDP. The absence of electrophysiological or histological evidence consistent with CIDP does not preclude the diagnosis. Similar caution should be exercised when interpreting demyelinating characteristics in nerve conduction studies or nerve biopsies performed on patients without clinical evidence of CIDP. CIDP has a variable prognosis like multiple sclerosis due to its heterogeneity. On average, 20-65% of patients experience a relapsing and remitting course, and the remainder experience a progressive course [29]. Most of the individuals with CIDP who do not have comorbid diseases respond to treatment, even more so when their CSF protein level is elevated.
The objective of this case report is to give essential information about the occurrence of new-onset CIDP, its clinical course, and diagnostic data that may be relevant in the management of comparable post-COVID-19 vaccination presentations. The progression of this patient's symptoms (both motor and sensory) was far more rapid than would be expected with diabetic polyneuropathy. Her symptoms were a bit too symmetric for diabetic amyotrophy. Around 90% of patients with CIDP have a high CSF protein level (more than 45 mg/dL) without CSF pleocytosis [30][31][32], which differentiates CIDP from multifocal motor neuropathy (MMN), which has a normal CSF protein level [33]. Albuminocytologic dissociation verifies the presence of immune-mediated polyneuropathy. Our diagnosis was further substantiated by nerve conduction studies and electromyography findings. The diagnosis of subacute GBS was considered in the differential. However, characteristics such as the lack of a distinct onset of symptoms and lack of autonomic symptoms went against the possibility of subacute GBS. Additionally, the patient showed symptoms three months after receiving the second dose of COVID-19 vaccination. The symptoms peaked sometime during the fifth month following their initiation. This goes against GBS, where symptoms nadir around eight weeks or less.
In our case, the relationship between the second dose of the mRNA-1273 (Moderna) vaccination and the onset of neuropathy is very evident, implying that the vaccine may have a triggering effect. This case report contributes to the limited body of evidence suggesting that immunization may contribute to the etiology of CIDP by providing one of the first detailed accounts of CIDP provoked by the Moderna vaccine. A close temporal link between vaccination, symptom onset, and a thorough diagnostic work-up to rule out other possible causes met the WHO criteria for identifying the causality of an adverse event following immunization on an individual basis. This led to the conclusion that this adverse event was most likely caused by the immunization.

Conclusions
Our observations, combined with those of earlier case reports, indicate that COVID-19 vaccinations may be associated with inflammatory neuropathies. Awareness of this uncommon but possible adverse effect is critical for prompt diagnosis and treatment. The occurrence of CIDP described in this case coincided with the Moderna COVID-19 vaccine. More research is needed to determine whether our patient's clinical presentation was just a coincidence or if there is a cause-and-effect relationship. This is the first reported case of CIDP, which developed post-mRNA-1273 (Moderna) vaccination outside of very few reports in VAERS. At this point, the true link between this case and COVID-19 vaccination remains uncertain. Further work will be required in the future to ascertain a true association between COVID-19 vaccinations and CIDP rather than a mere coincidental finding. When comparing the incidence of CIDP following vaccination to the incidence of CIDP occurring spontaneously, large observational studies will be required to establish a causal relationship. As we learn more about the long-term consequences of COVID-19 vaccines, adding any such information to clinical databases and research is incredibly valuable.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.